Hepatocellular carcinoma (HCC) is among the fastest-growing causes of cancer-related death in the U.S due to a lack of early detection strategies and therapies that target this highly heterogeneous tumor. Alcohol, a frequently abused recreational drug and hepatitis C virus (HCV), which establishes chronic infection, cause liver damage and cirrhosis and dramatically enhance risk for HCC. While great strides have been made in treating HCV infection, most individuals do not know they are infected. Individuals cured of HCV or who break their alcohol addiction remain at long-term risk for HCC. While screening liver cirrhosis patients for HCC clearly reduces mortality, >50% of HCCs are negative for the one blood-based marker available (AFP), emphasizing the dire need for better screens. Thus, it is crucial to gain a complete understanding of the pathological processes driving HCC in order to define those at greatest risk for HCC and develop new therapies to prevent or reverse liver carcinogenesis. While evidence that alcohol and HCV induce genetic changes is limited, we and others have shown that components of the epigenome, particularly DNA methylation (5mC) and DNA hydroxymethylation (5hmC), are profoundly disrupted during liver disease progression. One of the greatest challenges, however, is determining which epigenetic changes are drivers and which represent novel HCC early detection markers. We comprehensively examined genome-wide 5mC/5hmC analyses in HCC to reveal high frequency etiologic- and disease-stage specific changes, and we identified a panel of putative epigenetic drivers that we build on in this proposal. Based on these data, we propose three aims to test the central hypothesis of this application that epigenetic heterogeneity in cirrhosis underpins HCC development and progression. Elucidating the mechanisms underlying this effect is expected to yield novel drivers of HCC and early detection markers. Given that these are epigenetic targets, they also represent clinically targetable drivers. In aim 1 we define epigenetic heterogeneity during carcinogenesis of premalignant liver cirrhosis to HCC and define epigenetic drivers of tumor evolution. In aim 2, building on preliminary and published data identifying epigenetic driver loci and developing novel cell culture and PDX models, we functionally interrogate the role of loci targeted by aberrant 5mC/5hmC in driving HCC. Finally, in aim 3 we identify circulating cell-free DNA methylation signatures that differentiate between patients with cirrhosis and patients with cirrhosis and HCC and study their relationships with tumor heterogeneity. Results from these studies are expected to greatly expand our understanding of how epigenetic changes drive HCC, provide new actionable targets for treatment, and establish the foundations for future studies developing 5mC as an HCC early detection marker.